Description: (Verbatim from the applicant's abstract) The overall objective of the proposed study is to advance our initial work on Coherent Anti-Stokes Raman Scattering (CARS) microscopy to the next level: imaging of single macromolecules, biological membranes, and living cells using vibrational spectroscopy. The specific aims of the proposed studies are: 1. To improve the sensitivity of CARS microscopy to detect single macromolecules, such as proteins or DNA fragments. The nonresonant contributions from the solution or substrate limit CARS sensitivity. We propose to use time-resolved CARS to remove the nonresonant contributions completely. This will greatly enhance the sensitivity and make CARS detection of single protein and DNA molecules possible. 2. To conduct intracellular imaging of chromosomes, microtubules, and actin filaments. In order to identify protein and DNA spectroscopically, we need high spectral resolution (about 5 cm(-1)). We propose to use a new laser system to achieve this high spectral resolution. This will allow us to image and distinguish microtubules and actin filaments by CARS contrast. We will use this approach to study the structure and dynamics of cytoskeletons and chromosomes. 3. To develop high-resolution near-field CARS microscopy for imaging individual protein molecules in biological membranes. We will combine CARS with high-resolution near-field microscopy using metal tips, a technique we recently developed. This will allow us to image individual protein molecules in biological membranes with 10-20 nm resolution. This proposal focuses on the development of a new methodology to tackle compelling problems in biology. Although we give many examples of anticipated applications, the new methodology will likely to lead to exciting unexpected results.